VACCINE STUDIES: A crucial element in the development of effective prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model. It induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Therefore, candidate vaccines can be evaluated not only for their ability to prevent infection but also for their ability to prevent AIDS. The major vaccine effort within the laboratory has been an evaluation of the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector, based upon a pilot study of 12 macaques in which animals immunized with the MVA-SIV recombinant exhibited significant reduction in viremia and prolonged survival. Three recombinant MVA viruses were generated that expressed either gag-pol alone, env alone, or a combination of gag-pol and env. A cohort of 24 rhesus macaques were immunized (six per immunogen) to evaluate immunogenicity and protective efficacy after intravenous challenge. Four sequential immunizations resulted in moderate boosting of both gag and env-specific antibody responses but none of the animals developed neutralizing antibody specific for the challenge strain. All macaques became infected following intravenous challenge with SIVsmE660. However, plasma viremia in each of the groups immunized with MVA-SIV recombinants was significantly reduced (p=0.03) as compared to the group vaccinated with nonrecombinant MVA. There was no significant difference in viral load between the groups immunized with MVA-SIV recombinants. These data demonstrate that vaccination with MVA-SIV recombinants results in significant protection from high viremia and AIDS presumably mediated through cell mediated immunity. Evaluation of gag-specific CTL was performed in collaboration with Dr. Norman Letvin, utilizing macaques expressing the MamuA*01 MHC Class I haplotype for which a dominant gag-specific epitope (p11 C) has been identified. Four MamuA*01+ macaques were immunized with the MVA-gag-pol recombinant virus at 0 and 13 weeks and an additional two macaques were immunized with MVA and sequential generation of a p11C-specific CTL response was measured utilizing both traditional CTL assays as well as MHC Class I/Peptide tretramer staining. Macaques immunized with MVA expressing gag-pol developed CD8+ CTL as early as 2 weeks after the first immunization (2 of 4) which was boosted in all animals to high levels after the second immunization. Challenge of these macaques with SIVsmE660 resulted in a rapid and substantial anamnestic CTL response. Viral load set point was reduced in the macaques immunized with MVA-Gag-pol as compared to controls, however, the difference was not statistically significant. However, much of the spread in viremia in the immunized macaques could be explained by their response to immunization since there was an inverse correlation between the magnitude of the CTL response during vaccination and plasma viral load set point established after SIV challenge. Finally, we evaluated the MVA strategy using a different challenge model, SHIV/89.6P infection of rhesus macaques to allow a more direct comparison with results of other vaccine strategies. Four MamuA*01+ macaques were immmunized with MVA expressing SIVmac239 gag-pol and MVA expressing HIV/89.6 env sequentially three times and CTL responses were monitored by teramer staining for the dominant Gag p11C epitope and the subdominant Env p41A epitope. All animals mounted a c11C response whereas only one animal mounted a detectable response to the subdominant env epitope. This contrasts with a uniform response to both epitopes in macaques immunized with DNA. Following challenge with pathogenic SHIV/89.6P a significant reduction in set point viremia and partial protection from CD4 lymphocyte depletion was observed when compared with animals immunized with MVA nonrecombinant. This degree of protection is only slightly less robust than that observed in macaques immunized with cytokine augmented DNA (Letvin lab) and DNA prime-MVA boosted macaques (Robinson and Moss lab). The ability to control SHIV89.6 infection demonstrates the usefulness and effectiveness of this vaccine strategy as a sole immunogen or as part of a prime-boost strategy. Following challenge, a substantial expansion of CTL specific for the subdominant env peptide was observed despite the fact that this epitope was not recognized pre-challenge in these animals. PHYLOGENY OF SIV/HIV: The human immunodeficiency viruses, HIV-1 and HIV-2, are members of an extensive family of primate lentiviruses that appear to have their origins in African primates. Each of the human viruses presumably arose relatively recently following cross-species transmission from a naturally-infected primate to humans. In the case of HIV-2, the precursor appears to be SIVsm from sooty mangabey monkeys (Cercebus atys) whereas the origins of HIV-1 arose from SIVcpz from chimpanzees. The goal of this portion of the project is to molecularly characterize novel SIV isolates from wild-caught African monkeys more extensively. We initiated these studies by characterizing SIV isolates from three of the species of African green monkeys (vervets, grivets and tantalus), then SIV from Sykes monkeys (C. albogularis) and most recently SIV from lhoest monkeys (C. lhoesti). We also characterized SIV from another related monkey species, SIVsun isolated from a suntailed monkey. SIVsun clusters phylogenetically with SIVlhoest establishing the species-specific evolution of this lineage. These viruses also cluster with SIVmnd from mandrills although the genetic relationship is quite distant. Studies in the Peeters' lab have recently revealed that mandrills are infected with two distinct types of SIVs, one being the previously characterized virus now named SIVmnd-1 which is related to virus of the SIVlhoest species. The other appears to be a recombinant between SIVmnd-1 and SIV from drills (SIVdrl). Both SIVmnd-2 and SIVdrl have regions of homology with SIV from redcapped mangabeys (SIVrcm) but neither SIVrcm nor SIVdrl have been fully characterized. Recently we cloned and sequenced the entire genome of SIVrcm isolated from a wild caught redcapped mangabey from Nigeria and demonstrated that this virus is a complex recombinant. Comparison of SIVmnd-2 with SIVrcm revealed that SIVmnd-2 was a mosaic genome, highly related to SIVrcm in the 5' portion of the genome and to SIVmnd-1 in the 3' portion of the genome. Thus it appears that SIVrcm while in itself a recombinant is also the source of a portion of the SIVmnd-2 genome. These data are suggestive of cross-species transmission and recombination in the evolution of these viruses. Finally we characterized a SIVdrl and novel SIVmnd-type 2 isolate. These studies revealed that both of these viruses are recombinants between a SIVrcm-like virus and a SIVlhoest like virus and share a common recombination point. These data are consistent with a complex history of recombination and cross-species transmission involving SIV in drills, mandrills, redcapped mangabeys and L'hoest monkeys.